Volume 121, Issue 6, Pages 1380-1390 (December 2001) Human pancreatic acinar cells lack functional responses to cholecystokinin and gastrin  Baoan Ji, *, Yan Bi, *, Diane Simeone, ‡, Richard M. Mortensen, *, Craig D. Logsdon, *  Gastroenterology  Volume 121, Issue 6, Pages 1380-1390 (December 2001) DOI: 10.1053/gast.2001.29557 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Isolated human pancreatic acini appear intact and well polarized. Acini were prepared from human pancreas and viewed with an inverted-phase microscope. Cells show few signs of stress and appear to have normal cellular polarity with abundant apically appearing granules. Gastroenterology 2001 121, 1380-1390DOI: (10.1053/gast.2001.29557) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Human pancreatic acini secrete amylase in response to carbachol but not to CCK-8 or gastrin. Isolated human pancreatic acini, either uninfected or infected for 4 hours with an adenovirus expressing the human CCK-B receptor, were incubated with increasing concentrations of CCK-8, gastrin, or carbachol at 37°C for 30 minutes. The concentration of amylase released into the medium was measured using colorimetric reagent and was expressed as a percentage of initial acinar amylase content. Data are means of 3 separate experiments. Gastroenterology 2001 121, 1380-1390DOI: (10.1053/gast.2001.29557) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Human pancreatic acini have increased levels of intracellular Ca2+ in response to carbachol but not to CCK-8. Measurement of emitted fluorescence allowing an estimation of intracellular Ca2+ was performed with Fura 2–loaded acini using an Attofluor digital imaging system. At the times indicated, CCK-8 (100 nmol/L) or carbachol (1 mmol/L) was introduced. (A) Freshly prepared acini were treated with CCK-8 and then carbachol. (B) Acini that were infected with the adenovirus bearing human CCK-B receptor for 4 hours were treated with CCK-8 (100 nmol/L). (C) Acini that were infected with the adenovirus bearing rat CCK-A receptor for 4 hours were treated with CCK-8 (100 nmol/L). Data shown are typical and were representative of 4 separate experiments. Gastroenterology 2001 121, 1380-1390DOI: (10.1053/gast.2001.29557) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 ERK phosphorylation is stimulated in human pancreatic acini by carbachol but not CCK-8. To detect the phosphorylation of ERKs, acini were treated with CCK-8 (100 nmol/L) or carbachol (1 mmol/L) for 15 minutes then sonicated in lysis buffer. (A) Representative blots from a typical experiment. (B) Autoradiographs from 3 independent experiments were optically scanned, and the density of the bands was quantitated using the Molecular Analyst/PC image analysis software (Bio-Rad Laboratories). Results are means ± SE. *P < 0.05 compared with control. Gastroenterology 2001 121, 1380-1390DOI: (10.1053/gast.2001.29557) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 RT-PCR amplifies both CCK-A and CCK-B receptor mRNA from total RNA prepared from whole pancreas and isolated acini. RNA was prepared from pancreatic acini and from whole pancreas. RT-PCR was performed using 70 ng DNase-purified RNA; the results are representative of 3 independent experiments. That the bands amplified represented the expected genes was verified by sequencing. Gastroenterology 2001 121, 1380-1390DOI: (10.1053/gast.2001.29557) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Quantitative real-time RT-PCR indicates that CCK receptor mRNAs are expressed at low levels in human acinar cells. Real-time quantitative RT-PCR was conducted using SYBR green I to monitor the PCR products with an I-Cycler real RT-PCR detection system. (A) For calculation of the starting quantity of each mRNA in the samples, standard curves were prepared using serially diluted plasmids bearing CCK-A, CCK-B, or m3 receptor cDNAs. Data from representative standard curves are shown, indicating the cycle at which the signal reached the threshold for several dilutions of each plasmid. (B) Quantitation of mRNA expression was conducted using standard curves for human CCK-A, CCK-B, and m3 Ach receptors and using 65 pg of total RNA per acinar cell20 as an estimate of the cell numbers. (C) Amplification of RNA samples prepared from whole pancreas and isolated acini were compared to determine whether islets were a major source of receptor mRNA. Insulin mRNA was used as a control for detection of islet cell RNA contamination in the RNA prepared from isolated acini. The relative expression was determined by comparing the number of cycles required to reach threshold in RNA prepared from whole pancreas and isolated acini. The fold acinar mRNA level was calculated as 2(acini threshold − pancreas threshold). Data were from 4 independent runs comparing whole pancreas and isolated acini and are expressed as means ± SE. Gastroenterology 2001 121, 1380-1390DOI: (10.1053/gast.2001.29557) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 In situ hybridization detects m3 Ach receptor and insulin mRNA but not CCK receptor mRNA in human pancreas. In situ hybridization was performed on 10-μm frozen sections of human pancreas using digoxigenin-labeled RNA probes for (A, B) CCK-AR, (C, D) CCK-BR, (E, F) m3 AchR, and (G, H) insulin. Sense probes (A, C, E, G) were used as negative controls. After incubation with the labeled probes, the sections were incubated with an antidigoxigenin antibody conjugated with alkaline phosphatase and detected by the formation of a dark precipitate. No specific localization was observed for CCK-A or CCK-B receptor mRNAs, whereas m3 AchR mRNA was observed throughout the acinar portion of the pancreas and insulin mRNA was observed localized to islets (arrow). Gastroenterology 2001 121, 1380-1390DOI: (10.1053/gast.2001.29557) Copyright © 2001 American Gastroenterological Association Terms and Conditions